Viral fitness predicts the magnitude and direction of perturbations in the infected host transcriptome

Héctor Cervera; Silvia Ambrós; Guillermo P. Bernet; Guillermo Rodrigo; Santiago F. Elena

Virus genotypes and plant inoculations. The infectious clone pMTEV contains a full copy of the genome of a WT TEV strain isolated from tobacco ( Fig. 1A ; GenBank accession DQ986288) 69 . Six TEV mutant genotypes were constructed by site-directed mutagenesis starting from template plasmid pMTEV as described in ref. 70 (mutants AS13, CLA2 and CLA11) and ref. 32 (mutants PC48, PC55 and PC95). Table 1 shows the characteristics of the seven genotypes used in the study. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/206789 doi: bioRxiv preprint RNA preparations. RNA extraction from 100 mg of fresh tissue per plant was performed using Agilent Plant RNA Isolation Mini Kit (Agilent Technologies) following the manufacturer's instructions. The concentration of total plant RNA extract was adjusted to 50 ng/µL for each sample. Each RNA sample was re-sequenced again at this stage to ensure the constancy of the genotypes as described above. Fitness evaluations. Total RNA was extracted and virus accumulation quantified by RT-qPCR as described above and detailed previously 72 . Virus accumulation (expressed as genomes/ng of total RNA) was quantified 8 dpi for all genotypes with the exception of AS13, that was quantified 15 dpi. These sampling times assure that viral populations were at a quasi-stationary plateau in N. tabacum. These values were then used to compute the fitness of the mutant genotypes relative to that of the WT genotype using the expression W = (R t ⁄R 0 ) 1/t , where R 0 and R t are the ratios of accumulations estimated for the mutant and WT viruses at inoculation and after t days of growth, respectively 32 .